The colon has a key physiologic role in salt and water conservation, mediated in large part by colonic sodium absorption. Pro-absorptive compounds like luminal short-chain fatty acids (SCFAs) physiologically stimulate sodium absorption by activating apical Na+/H+ exchange in colonocytes, and effectively combat diarrheal disease. Our long-term objective is to define cellular mechanisms regulating colonic apical Na+/H+ exchangers. In two model systems, mouse distal colonic epithelium and HT29-Cl cells (a cloned human colon carcinoma cell line), results tentatively suggest NHE2 is the apical Na+/H+ exchanger isoform activated by luminal SCFAs. Experiments also build on prior evidence that SCFAs activate Na+/H+ exchange via pH microdomains in and around colonocytes. We will primarily use confocal and two-photon microscopy to measure subcellular events in colonic mucosa and polarized epithelial cells. The first specific aim asks whether NHE2 is the apical Na+/H+ exchanger in the colonic crypt. Normal and NHE2 knockout mice will be used in conjunction with isoform specific NHE inhibitors to define the distribution of apical Na+/H+ exchange along the crypt-to-surface axis after activation by SCFA. We will test if diminished NHE2 contributes to the NHE3 knockout mouse phenotype in distal colon. The second aim asks if NHE2 is regulated by translocation from the plasma membrane. We will ask if the endogenous apical Na+/H+ exchanger of crypt colonocytes or HT29-C1 cells has a kinetic response (deltaVmax) consistent with membrane translocation from the plasma membrane. Confocal imaging of NHE2-CFP chimeric protein (CFP a pH-insensitive fluorescent protein) in transfected PS 120 fibroblasts or HT29-C1 cells will be used to visualize subcellular distribution of NHE2. We will ask if membrane translocation occurs in response to SCFA, or effectors known to change Vmax of NHE2 (ATP-depletion, hyperosmolarity). The third aim will discover how pH is regulated directly adjacent to NHE2. NHE2-YFP chimeras (YFP is a pH-sensitive fluorescent protein) have a built-in pH sensor, which can report on the NHE transport activity from the environment directly adjacent to the protein. We will use confocal microscopy to measure this "near-NHE" pH in parallel with measurements of cytosolic pH, using PS120 or HT29- C1 cells transfected with the constructs. We will ask (1) how near-NHE2 pH regulation is affected by being near the membrane versus near a proton transporter, and (2) if near-NHE2 pH values change interpretation about effects of regulatory agonists or intracellular proton activation kinetics. To translate these results to native tissue, experiments will explore the factors that limit generation of intracellular pH gradients in crypt colonocytes, and create NHE2-YFP transgenic mice to initiate exploration of near-NHE pH in mouse.